Dimerization of HIV-1 protease (PR) subunits is an essential process for PR's acquisition of proteolytic activity, which plays a critical role in the maturation of HIV-1. Thus, it is thought that the disruption of the dimerization process can inhibit HIV-1 maturation. In this regard, DRV has been shown to effectively disrupt the dimerization of PR monomer subunits as determined by the fluorescence resonance energy transfer (FRET)-based HIV-1 expression assay. We thus determined whether GRL-142 exerted PR dimerization inhibition activity. In the absence of compounds, the mean CFPA/B ratio obtained was 1.16, indicating that PR dimerization clearly occurred. However, the ratio was decreased to 0.91 in the presence of 100 nM DRV, demonstrating that DRV blocked the dimerization of the PR subunit. Interestingly, GRL-142 blocked the dimerization at much lower concentrations (0.1 nM) than DRV. These data strongly suggest that as in the case of DRV, GRL-142 has bimodal HIV-1 inhibition mechanism, (i) inhibition of HIV-1 PR dimerization and (ii) inhibition of enzymatic activity of PR. We have previously demonstrated that PR monomer subunits initially interact at the active site interface, generating unstably dimerized PR subunits, and subsequently the termini interface interactions occur, completing the dimerization process. DRV binds in the proximity of the active site interface of PR and blocks PR subunits dimerization. Therefore, we asked whether GRL-142 binds to monomer subunits using electrospray ionization mass spectrometry (ESI-MS). As shown in Figure 2C, the ESI-MS spectra of PR containing D25N substitution (PRD25N), which was folded in the presence of drugs revealed five peaks of differently charged ions in the range of mass/charge ratio (m/z) of 1,500-2,900. Since +5 charged monomer ion and +10 charged dimer ion have the same m/z (m/z=2164.75 for PRD25N), the greatest peak detected at m/z 2164.75 was determined to represent two forms, a PR monomer and PR dimer. Thus, the five peaks represent a monomer, two dimers, and two monomer+dimers. When unfolded PRD25N was re-folded in the presence of DRV, six additional significant peaks appeared, three for monomer+DRV, and three for dimer+DRV. When the same PRD25N was re-folded in the presence of GRL-142, six significant peaks appeared, representing three for monomer+GRL-142, and three for dimer+GRL-142. Each of the six additional peaks seen with GRL-142 appeared greater than those seen with DRV. When compared with the heights of the dimer+monomer peak rendered 1.0, the average height of the three peaks of DRV-bound monomers and that of the three peaks of GRL-142-bound monomers were 0.046 and 0.312, respectively; and the average height of the three peaks with DRV-bound dimers and that of the three peaks with GRL-142-bound dimers were 0.060 and 0.188, respectively. These data suggest that GRL-142 more tightly bound to monomers by 6.78-fold and to dimers by 3.13-fold than DRV and at least in part explain the reason GRL-142 much more strongly blocked PR dimerization than DRV. We also examined the thermal stability of PRD25N in the presence of saquinavir (SQV), DRV, or GRL-142, using differential scanning fluorimetry (DSF). As illustrated in Figure 2D, the Tm value of PRD25N alone was 54.92C, while in the presence of SQV and DRV, the values increased to 58.14C and 58.21C, respectively, suggesting that the thermal stability of PRD25N increased when SQV and DRV bound to PRD25N. However, in the presence of GRL-142, the Tm value of PRD25N turned out to be substantially high at 65.65C and the difference in Tm values between PRD25N alone and GRL-142-bound PRD25N reached as high as 10.73C, suggesting that GRL-142 more strongly binds to PRD25N than SQV or DRV. When we asked whether DRV and GRL-142 bound to TFR-PRD25N-7AA-His6, a His-tagged transframe precursor form of PRD25N that contains seven N terminus amino acids of reverse transcriptase (7AA; PISPIET), both DRV and GRL-142 clearly bound to TFR-PRD25N-7AA-His6, although the binding of GRL-142 (52.07C) appeared to be significantly greater than that of DRV (43.30C). Considering that GRL-142 much more strongly binds to PR monomer subunits than DRV, as in the case of DRV, GRL-142's monomer binding should be involved in the Gag-Pol auto-processing inhibition and even more effective than that of DRV.